Neurobiology of Stress

Stress responses are essential for coping with immediate threats and maintaining physiological homeostasis. While acute stress activates adaptive neuroendocrine and behavioral mechanisms, chronic stress leads to desensitization of these responses, disrupting hormone secretion, neuronal activity, and behavior. Chronic stress is a well-established risk factor for neuropsychiatric disorders, many of which show distinct prevalence and presentation patterns between sexes. However, the neurobiological mechanisms underlying these sex-dependent effects remain poorly understood. This study investigated how acute and chronic stress differentially affect neural activation patterns in male and female mice, with the hypothesis that sex-specific adaptations to chronic stress underlie divergent vulnerabilities to neuropsychiatric disorders. We employed three experimental groups: a control group (no stress), an acute stress group (one hour of restraint stress), and a chronic stress group (one hour of restraint stress daily for ten days). Neural activity was assessed by quantifying c-Fos-positive cells using immunohistochemistry. Acute stress induced widespread neural activation in both sexes, with notable sex differences in c-Fos expression in regions of the hypothalamus, amygdala, and midbrain. Chronic stress led to the desensitization of neuronal activity in most of these regions. Notably, chronically stressed females exhibited more desensitization in specific hypothalamic and amygdaloid regions compared to males. Despite this, corticosterone release remained elevated in stressed females, indicating a decoupling of hormonal and neural responses. These findings suggest that chronic stress elicits distinct neural adaptations in males and females, potentially contributing to the sex-specific vulnerability to neuropsychiatric disorders. Understanding these mechanisms may inform targeted interventions for stress-related pathologies.

A single, severe episode of stress can bring about myriad responses amongst individuals, ranging from cognitive enhancement to debilitating and persistent anxiety; however, the biological mechanisms that contribute to resilience versus susceptibility to stress are poorly understood. The dentate gyrus (DG) of the hippocampus and the basolateral nucleus of the amygdala (BLA) are key limbic regions that are susceptible to the neural and hormonal effects of stress. Previous work has also shown that these regions contribute to individual variability in stress responses; however, the molecular mechanisms underlying the role of these regions in susceptibility and resilience are unknown. In this study, we profiled the transcriptomic signatures of the DG and BLA of rats with divergent behavioral outcomes after a single, severe stressor. We subjected rats to three hours of immobilization with exposure to fox urine and conducted a behavioral battery one week after stress to identify animals that showed persistent, high anxiety-like behavior. We then conducted bulk RNA sequencing of the DG and BLA from susceptible, resilient, and unexposed control rats. Differential gene expression analyses revealed that the molecular signatures separating each of the three groups were distinct and non-overlapping between the DG and BLA. In the amygdala, key genes associated with insulin and hormonal signaling corresponded with vulnerability. Specifically, Inhbb, Rab31, and Ncoa3 were upregulated in the amygdala of stress-susceptible animals compared to resilient animals. In the hippocampus, increased expression of Cartpt - which encodes a key neuropeptide involved in reward, reinforcement, and stress responses - was strongly correlated with vulnerability to anxiety-like behavior. However, few other genes distinguished stress-susceptible animals from control animals, while a larger number of genes separated stress-resilient animals from control and stress-susceptible animals. Of these, Rnf112, Tbx19, and UBALD1 distinguished resilient animals from both control and susceptible animals and were downregulated in resilience, suggesting that an active molecular response in the hippocampus facilitates protection from the long-term consequences of severe stress. These results provide novel insight into the mechanisms that bring about individual variability in the behavioral responses to stress and provide new targets for the advancement of therapies for stress-induced neuropsychiatric disorders.

Stress is an important risk factor for the development of psychiatric disorders and men and women tend to react differently to stress. Sex differences are also observed in many stress-related psychiatric disorders such as depression, anxiety disorders or addiction. Therefore, identifying specific neuroadaptations induced by stress, in males and females, is a necessary step to the understanding of stress-related sex dimorphism in these disorders. Here, we tested the hypotheses that acute stress could affect plasticity in the anterior insular cortex (aIC)-nucleus accumbens core (NAcC) pathway, two structures involved in the stress response, in a sex-dependent manner. Using in vivo extracellular recordings in anesthetized rats, we show that synaptic plasticity in the aIC-NAcC pathway is different between male and female rats. Whereas in males, long-term potentiation and long-term depression were equally induced, in females, there was mostly a long-term potentiation induced. Moreover, stress affected synaptic plasticity in the aIC-NAcC differently in male and female rats. In males, stress induced a loss of long-term-depression that lasted for at least 24h, whereas in females, stress induced less neurons displaying LTP, which did not last. These results demonstrate that integration of aIC information to NAcC is different between males and females. This study provides mechanistic support for differential reactivity to stress between males and females that may relate to stress-related psychiatric disorders and sex dimorphism in these disorders.

Acute stress triggers plasticity of forebrain synapses as well as behavioral changes. Here we reveal that Tumor Necrosis Factor (TNF) is a required downstream mediator of the stress response in mice, necessary for stress-induced synaptic potentiation in the ventral hippocampus and for an increase in anxiety-like behaviour. Acute stress is sufficient to activate microglia, triggering the long-term release TNF. Critically, on-going TNF signaling in the ventral hippocampus is necessary to sustain both the stress-induced synaptic and behavioral changes, as these could be reversed hours after induction by antagonizing TNF signaling. This demonstrates that TNF maintains the synaptic and behavioral stress response in vivo, making TNF a potential novel therapeutic target for stress disorders.

Combined oral contraceptives (OCs), containing synthetic estrogen and a progestin such as levonorgestrel (LVNG), are widely used, and up to 10% of users experience adverse mood states and increased depression risk. It is well-established that OCs modulate the hypothalamic-pituitary-adrenal (HPA) axis and blunt the cortisol responses to acute stress. This interaction with stress regulatory pathways is one mechanism by which OCs might impact mood. Here, we used a mouse model of OC exposure (ethinyl estradiol (EE) + LVNG) to investigate how OCs affect regulation of the diurnal CORT cycle and stress-related signaling in the dorsal and ventral hippocampus and paraventricular nucleus of the hypothalamus (PVN). We found that EE+LVNG did not alter basal corticosterone (CORT) levels, but impaired glucocorticoid receptor (GR) - mediated negative feedback in the dexamethasone suppression test. Molecular analyses revealed distinct, region-specific effects. In the dorsal hippocampus, EE+LVNG enhanced glucocorticoid receptor (GR)-dependent gene signaling and prolonged Fkbp5 induction. In the ventral hippocampus, EE+LVNG enhanced mineralocorticoid receptor (MR)-dependent signaling and reduced stress-induced corticotropin-releasing factor expression. In the PVN, EE+LVNG reduced MR expression and modulated MR-dependent signaling. Together, these findings demonstrate that chronic OC exposure disrupts GR- and MR-dependent regulation across stress-related brain regions and impairs glucocorticoid feedback, providing potential mechanisms by which OCs blunt stress responsivity, modify long-term HPA-axis function, and increase susceptibility or resilience to stress and depression.

The lateral habenula (LHb) inhibits midbrain monoaminergic neurons, thereby regulating emotion/cognition. Abnormally high activity in the LHb causes behavioral disorders, but how stressful experiences affect neuronal circuits underlying emotion remains poorly understood. Here, we report the effects of chronic stress on the LHb in postnatal day (P)1-9, P10-20, and P36-45 mice in the pre-, early, and late stages of LHb maturation. At P60, only mice exposed during P10-20 exhibited LHb-specific changes: abnormally high-stress reactivity shown by the expression of the immediate-early gene product (Zif268/Egr1) with insufficient number of parvalbumin (PV) neurons containing GABA. Furthermore, these mice showed anxiety/depression-like behaviors in the light-dark box test/forced swim test. Thus, experiences in early-life are essential for the maturation of neuronal circuits underlying emotion. Early-life stress is thought to have caused anxiety/depression in adulthood by disrupting the maturation of inhibitory PV neurons in the LHb in a period-specific manner.

Repeated social stress is a significant factor in triggering depression in vulnerable individuals, and genetic and environmental factors interact to contribute to this vulnerability. Interestingly, the role of experience in shaping vulnerability is not well studied. To what extent does an individuals initial reaction to a given stressor influence their response to similar stressors in the future? And how is this initial response encoded at the neural level to bias towards future susceptibility or resilience? The Chronic Social Defeat Stress (CSDS) mouse model offers an ideal opportunity to address these questions. Following 10 days of repeated social defeat, mice diverge into two distinct populations of social reactivity: resilient (interactive) and susceptible (avoidant). It is notable that the CSDS paradigm traditionally uses genetically inbred mice, indicating that this divergence is not genetically determined. Furthermore, the emergence of the two phenotypes only occurs following several days of exposure to stress, suggesting that the repeated experience of social defeat influences future susceptibility or resilience. In this study, we asked whether specific patterns of neural activation during the initial exposure to the social defeat stress can predict whether an individual will eventually emerge as resilient or susceptible. To address this question, we used Fos-TRAP2 mouse technology to capture brain-wide neural activation patterns elicited during the initial stress exposure, while allowing the mice to go on to experience the full course of CSDS and diverge into resilient and susceptible populations. Using a high-throughput brain-wide cell counting approach, we identified the bed nucleus of the stria terminalis and lateral septal nucleus as key hubs for encoding social defeat. We also identified the basomedial amygdala as a hub for encoding future susceptibility, and the hippocampal CA1 area and medial habenula for encoding future resilience. Our findings demonstrate that the initial experience with social stress induces a distinct brain-wide pattern of neural activation associated with defeat, as well as unique activation patterns that appear to set the stage for future resilience or susceptibility. This highly orchestrated response to defeat is seen especially in animals that emerge as resilient compared to susceptible. Overall, our work represents a critical starting place for elucidating mechanisms whereby early experiences can shape vulnerability to affective disorders.

Chronic stress is a physiological state marked by dysregulation of the hypo-pituitary-adrenal axis and high circulating levels of stress hormones, such as corticosterone in mice or cortisol in humans. This dysregulated state may result in the development of mood disorders but the process by which this occurs is still unknown. The bed nucleus of the stria terminalis (BNST) serves as an integration center for stress signaling and is therefore likely an important area for the development of mood disorders. This project utilized a chronic variable mild stress (CVMS) paradigm to persistently stress mice for 6 weeks followed by RNA-Sequencing of the anterodorsal (ad) BNST and electrophysiology of corticotropin releasing hormone-expressing cells in the adBNST. Our results show significant sex-biases in the transcriptome of the adBNST as well as effects of CVMS on the transcriptome of the adBNST specifically in males. Female biased genes are related to synaptic transmission while male biased genes are related to RNA processing. Stress sensitive genes in males are related to synaptic transmission and synapse formation. Additionally, electrophysiology data showed that CVMS suppressed the M-current in males but not females. However, CVMS increased the strength of excitatory post-synaptic currents in females but not males. This suggests significant differences in how males and females process chronic stress. It also suggests that the BNST is more sensitive to chronic stress in males than in females.

Disruptions in excitatory-inhibitory (E/I) balance during neurodevelopment have been implicated in a range of psychiatric conditions, yet the neurochemical alterations associated to early-life insults and their potential contribution to E/I imbalance remain poorly understood. Using a "two-hit" rat model combining maternal immune activation (MIA; lipopolysaccharide -LPS- on gestational days 15-16) and peripubertal unpredictable stress (PUS; postnatal days 28-38), we examined the long-term effects of these insults, alone and in combination, on the adult dorsal hippocampus. Assessments included gene and/or protein expression of glutamatergic and GABAergic markers, endocannabinoid system enzymes, neuromodulatory amino acid level and prepulse inhibition (PPI) of the acoustic startle response. MIA increased GluN1 protein expression, while PUS reduced the Grin2a/Grin2b mRNA ratio, indicating incomplete NMDA receptor subunit maturation. GABA levels and GABA-A{gamma}2 expression were unchanged, suggesting deficient inhibitory compensation in the face of heightened excitatory tone. PUS increased Mgll gene expression, whereas a trend towards reduced Dagla expression was observed exclusively in non-stressed LPS-exposed animals, suggesting that MIA may suppress 2-AG synthesis only in the absence of subsequent stress. MIA and PUS displayed interactive effects on taurine levels, with elevation observed only in the double-hit condition; glycine was elevated by MIA independently of PUS. These findings support a model in which MIA and PUS converge on hippocampal E/I balance through complementary adaptations -- excitatory upregulation, incomplete synaptic maturation, and reduced endocannabinoid tone -- inadequately counterbalanced by inhibitory systems. Taurine and glycine emerge as potential markers of homeostatic compensation in response to early neurochemical dysregulation.

BackgroundChild adversity (CA), encompassing emotional, physical, and sexual maltreatment or abuse, affects a substantial number of children worldwide. Moreover, it is the leading predictor of psychiatric disorders such as major depressive disorder (MDD), anxiety, and suicidal behavior. Despite the robust link between CA and psychopathology, individual outcomes vary significantly, with some children demonstrating resilience. Resilience is an adaptive and dynamic process, which mitigates the long-term effects of CA, suggesting potential protective mechanisms that remain underexplored. This study investigates the role of the locus coeruleus-norepinephrine (LC-NE) system, a critical modulator of stress, cognition, and emotion, in mediating resilience and susceptibility following early life stress (ELS). MethodsUsing a maternal deprivation model combined with limited nesting and bedding, we examined behavioral, physiological, and neurobiological markers associated with ELS outcomes in mice of both sex. ResultsBehavioral clustering revealed distinct phenotypes: resilient, anxious, and depressive-like with sex-specific differences in distribution. Early markers, including body weight and ultrasonic vocalization (USV) patterns, predicted long-term susceptibility. Neuroanatomical analyses identified sex-specific LC-NE activation patterns associated with resilience and susceptibility, highlighting the caudal-dorsal LC as a critical region in males and females in different phenotypes, anxious in males and resilient in females. ConclusionThese findings highlight the impact of ELS on the LC-NE system and its role in shaping adaptive and maladaptive trajectories, offering insights into potential interventions targeting resilience mechanisms in children exposed to CA.

Background & AimsClinical evidence suggests significant interindividual differences in stress reactivity (SR), but biological mechanisms and therapeutic implications of these differences are poorly understood. We aimed to identify the biological basis of increased SR by investigating associations between a psychometric-based phenotype with blood transcriptomics profiles of increased sympathetic nervous system (SNS) activation and brain imaging phenotypes in irritable bowel syndrome (IBS) participants and healthy controls (HCs). MethodsA cross-sectional observational study design, transcriptomics profiling, multimodal brain imaging, and psychosocial assessments were obtained in 291 female and male IBS participants and HCs. Prior to analyses, unsupervised clustering was applied to derive high and low SR subgroups across participants based on two measures of SR. General linear models tested for SR group differences in clinical and biological parameters. Exploratory analyses examined associations between SR group-specific brain alterations and gene expression. ResultsThe high, compared to low SR group showed greater cyclic AMP response element-binding protein (CREB) gene expression consistent with tonic SNS activity and proinflammatory changes in whole blood. Brain imaging showed neuroplastic changes in the high SR group consistent with an upregulation of ascending arousal systems and sensory processing and integration regions, and functional connectivity changes in the central autonomic network. SR moderated the sex difference in extraintestinal symptoms. ConclusionsThe findings support a model of tonically increased SNS activity as a plausible risk factor for increased autonomic reactivity to psychosocial stressors and low grade immune activation in both IBS and HCs, with a greater prevalence in IBS. These findings may have important implications for personalized treatment interventions in IBS.

Consistent individual differences in behavior (e.g., personality types, stress coping styles) are a common occurrence across animal taxa. One hypothesis poses that the resulting constraints for within-individual behavioral variation may also lead to constraints for the evolution of behavior. With stress coping styles seen across taxa, it suggests a common underlying proximate mechanism. In this study we investigated neural activity patterns across the brain by quantifying immediate early gene expression in individuals with alternative stress coping styles in response to an acute stressor in zebrafish (Danio rerio). While immediate early gene expression levels of individual brain regions in the aversive brain network were similar across groups, functional network activity differed. There were several differences across groups including interactions between the basolateral amygdala and hippocampal homologs. One brain area that had many different connections across groups was the central gray. There were many differences involving central gray activity between proactive and reactive fish at baseline suggesting that baseline activity may prime for the reaction to stress. Collectively, baseline brain activity can predict behaviors, suggesting that these differences in brain interactions at baseline may be important for biasing behavioral responses to stressors that characterizes a stress coping style. Significance StatementIt is well known that individuals cope with stress in different ways but the underlying mechanisms are not well understood. In this study we investigate how different brain regions interact under stress in animals of different stress coping styles. We investigated activity patterns across several brain regions in fish with a passive or active response to stress. We used an innovative statistical method that allowed us to compare how the brain regions function together under stress. The central gray was the most different between proactive and reactive groups, suggesting that this region is important for biasing the response to stressors. Further baseline functional connectivity differed the most between groups, suggesting that activity at baseline is important for biasing the response to stress mainly through connections with the central gray. Ultimately, functional neural network activity better explains stress behaviors than individual brain activity.

Adolescence is a critical period of development with increased sensitivity toward psychological stressors. Many psychiatric conditions emerge during adolescence and animal studies have shown that that acute stress has long-term effects on hypothalamic pituitary adrenal axis function and behavior. We recently demonstrated that acute stress produces long-term electrophysiological changes in locus coeruleus and long-lasting anxiety-like behavior in adolescent male rats. Based on prior reports of increased stress sensitivity during adolescence and increased sensitivity of female locus coeruleus toward corticotropin releasing factor, we hypothesized that the same acute stressor would cause different behavioral and physiological responses in adolescent female and adult male rats one week after stressor exposure. In this study, we assessed age and sex differences in how an acute psychological stressor affects corticosterone release, anxiety-like behavior, and locus coeruleus physiology at short- and long-term intervals. All groups of animals responded to stress with elevated corticosterone levels at the acute time point. One week after stressor exposure, adolescent females showed decreased firing of locus coeruleus neurons upon current injection and increased exploratory behavior compared to controls. The results were in direct contrast to changes observed in adolescent males, which showed increased anxiety-like behavior and increased spontaneous and induced firing locus coeruleus neurons a week after stressor exposure. Adult males were both behaviorally and electrophysiologically resilient to the long-term effects of acute stress. Therefore, there may be a normal developmental trajectory for locus coeruleus neurons which promotes stress resilience in adults, but stressor exposure during adolescence perturbs their function. Furthermore, while locus coeruleus neurons are more sensitive to stressor exposure during adolescence, the effect varies between adolescent males and females. These findings suggest that endocrine, behavioral, and physiological responses to stress vary among animals of different age and sex, and therefore these variables should be taken into account when selecting models and designing experiments to investigate the effects of stress. These differences in animals may also allude to age and sex differences in the prevalence of various psychiatric illnesses within the human population.

Exposure to early life stress (ELS) can exert long-lasting impacts on emotional regulation. The corticolimbic system including the basolateral amygdala (BLA), ventral hippocampus (vHIP), and the medial prefrontal cortex (mPFC) plays a key role in fear learning. Using the limited bedding paradigm (LB), we examined the functional consequences of ELS on excitatory and inhibitory tone in the prelimbic (PL) mPFC after fear conditioning in rats. In adults, LB exposure enhanced in vivo glutamate release in the PL mPFC during fear conditioning in male, but not female offspring. In contrast, the glutamate response to fear conditioning was diminished in LB-exposed pre-adolescent males, but not females. We investigated whether reduced glutamatergic inputs and/or elevated inhibitory tone might contribute to the diminished glutamate response in the mPFC following LB in pre-adolescent male rats. Indeed, we found that LB exposure specifically increased the activation of PV, but not SST interneurons in layer V, but not layer II/III of the PL mPFC in fear-exposed pre-adolescent males. Presynaptic glutamate release probability was reduced by LB exposure in layer V, but increased in layer II/III of the PL mPFC. These functional changes might be related to the LB-induced alterations in the bilaminar distribution of BLA and vHIP projections to the PL mPFC we observed in pre-adolescent males. Overall, our findings suggest that ELS modifies glutamate release and PL mPFC function during fear conditioning in a sex- and age-dependent fashion, likely through layer-specific shifts in excitation/inhibition balance. Significance StatementEarly life stress (ELS) increases the risk of developing affective disorders and long-term emotional dysregulation might arise from disruptions in the development of the fear circuitry. This study examines how ELS modifies fear-induced activity of long-range excitatory projections and local inhibitory microcircuits in the developing prefrontal cortex. We tested whether ELS-induced alterations in prefrontal cortex function are sex- and age-dependent, leading to the well-documented sex differences in emotional behavioral outcome. Studying how ELS differentially modifies regional excitatory inputs and cell type specific activation in the prefrontal cortex during a critical period of brain development will enhance our understanding of the neurobiological mechanisms underlying the pathogenesis of emotional dysregulation and inspire more targeted intervention after exposure to early adversity.

Early life stress (ELS) events during sensitive postnatal time periods can recalibrate future stress responsiveness and precipitate mental disorders. Neurovascular adaptations can influence cognition, mood, and stress responses. Disruption of blood-brain barrier (BBB) integrity, which is formed by endothelial cells, astrocytes, and pericytes, has been implicated in affective disorders such as depression, which often arise from chronic stress experiences. Despite the BBB undergoing critical maturation stages during development, it remains poorly known how ELS influences brain vascular function, as previously shown for adult stress, and whether it augments BBB vulnerability to subsequent challenges. First, we took advantage of a public two-hit stress RNA-sequencing dataset and filtered for vascular enriched genes in the prefrontal cortex and nucleus accumbens, the two brain regions where BBB integrity is frequently compromised. This analysis revealed BBB-related gene ontology categories modulated by either ELS alone or its combination with adult stress. Then, using a mouse model combining ELS with chronic social defeat stress (CSDS) in adulthood, we found that ELS did not exacerbate CSDS susceptibility; instead, it increased social interactions and the likelihood of a resilient profile in both males and females. Transcriptomic profiling in our cohort further identified distinct sex- and region-specific BBB gene expression patterns associated with ELS and its interaction with CSDS. Additionally, we observed a reduction of corticosterone levels, the primary stress hormone, following CSDS. Altogether, these results indicate that ELS modulates stress responses when facing emotional challenges in adulthood, possibly through long-lasting changes of BBB function via the glucocorticoid system. HighlightsO_LIRNA-seq vascular filtering reveals BBB distinct ontology categories for ELS and AS C_LIO_LIELS increases the likelihood of a high social and resilient profile. C_LIO_LIPericytes gene expression associated to resilience is sex- and region-specific. C_LIO_LICORT response desensitizes after adult CSDS in both sexes. C_LI

Exposure to an acute stressor triggers a complex cascade of neurochemical events in the brain. However, deciphering their individual impact on stress-induced molecular changes remains a major challenge. Here we combine RNA-sequencing with selective pharmacological, chemogenetic and optogenetic manipulations to isolate the contribution of the locus coeruleus - noradrenaline (LC-NA) system to the acute stress response. We reveal that NA-release during stress exposure regulates a large and reproducible set of genes in the dorsal and ventral hippocampus via {beta}-adrenergic receptors. For a smaller subset of these genes, we show that NA release triggered by LC stimulation is sufficient to mimic the stress-induced transcriptional response. We observe these effects in both sexes, and independent of the pattern and frequency of LC activation. Using a retrograde optogenetic approach, we demonstrate that hippocampus-projecting LC neurons directly regulate hippocampal gene expression. Overall, a highly selective set of astrocyte-enriched genes emerges as key targets of LC-NA activation, most prominently several subunits of protein phosphatase 1 (Ppp1r3c, Ppp1r3d, Ppp1r3g) and type II iodothyronine deiodinase (Dio2). These results highlight the importance of astrocytic energy metabolism and thyroid hormone signaling in LC-mediated hippocampal function and offer new molecular targets for understanding how NA impacts brain function in health and disease.

Stress is a commonly reported seizure precipitant and may contribute to the development of psychiatric comorbidities in epilepsy, yet how chronic stress interacts with epileptic circuits remains poorly understood. We investigated the impact of chronic restraint stress on physiological, behavioral, and synaptic outcomes in a mouse model of Dravet syndrome, specifically corticotropin-releasing factor (CRF) neurons in the bed nucleus of the stria terminalis (BNST), a stress-responsive region implicated in epilepsy patients. Chronic restraint stress produced divergent hypothalamic-pituitary-adrenal axis responses, with stressed Dravet syndrome mice exhibiting elevated corticosterone, increased mortality in females, and increased locomotion and anxiety-like behavior. Ex vivo electrophysiological recordings revealed that chronic stress increased spontaneous excitatory event frequency onto BNST CRF neurons in both genotypes and selectively increased sEPSC and sIPSC amplitude in Dravet syndrome mice. Evoked recordings demonstrated genotype-specific effects of stress on glutamatergic transmission in CRF neurons of the DS group. This suggests greater stress-dependent remodeling of spontaneous and evoked synaptic activity in DS. These findings suggest chronic stress may worsen physiological and behavioral outcomes in Dravet syndrome and promote specific maladaptive alterations in BNST CRF circuitry. More broadly, these results suggest that stress interacts with seizure vulnerability and potentially contributes to neuropsychiatric comorbidities and epilepsy.

Stress, whether real or perceived, activates physiological and behavioral responses via the hypothalamic- pituitary-adrenal (HPA) axis and sympathetic nervous system activation. Under chronic stress, however, these adaptive responses become dysfunctional leading to pathological changes in behavior and health. Mitochondria are dynamic organelles essential for cellular energy production and for initiating glucocorticoid synthesis and release from adrenal glands during stress. Thus, mitochondria may represent a first line of response to environmental challenges. However, the effects of chronic stress on mitochondrial function within the HPA axis, particularly regarding sex differences, are unexplored. We exposed adult male and female C57BL6/J mice to four weeks of chronic unpredictable stress and examined behavioral and mitochondrial responses in the hypothalamus and adrenal glands - two key HPA axis regions. As previous reports indicated sex differences in stress responsivity, we hypothesized that chronic stress would differentially impact mitochondrial respiration within HPA axis regions in a sex-specific manner. Chronic stress increased avoidance behavior in males and passive coping behavior in females, indicating sex-specific behavioral responses. In females, stress significantly decreased mitochondrial respiration in both the hypothalamus and adrenal glands, while males were not significantly affected. In males, stress increased adrenal expression of mitochondrial complex II protein, which may have served a compensatory role to preserve mitochondrial function. Mitochondrial respiration significantly correlated with behavioral measures in stressed animals, highlighting a relationship between metabolism and stress-induced impairments. These findings reveal sex-specific metabolic adaptations to chronic stress and suggest that females may be more vulnerable to stress-induced mitochondrial dysfunction within the HPA axis. Clinical PerspectivesO_LIChronic stress is widely prevalent, associated with neuropsychiatric disease that affect women at a rate twice as high as men, and mediated by mitochondria, yet sex differences in the effects of chronic stress on mitochondrial function have not been characterized. C_LIO_LIDespite similar behavioral outcomes, chronic unpredictable stress exposure significantly decreases mitochondrial respiration only in the hypothalamus and adrenal glands from females, in association with stress-induced behavioral alterations. C_LIO_LIFemales may have increased vulnerability to metabolic effects of chronic stress and therapies targeting mitochondrial function may be more efficacious in preventing behavioral impacts of stress in females. C_LI

Identifying the neural circuits engaged and reshaped by chronic stress is critical for understanding how adaptive responses shift to maladaptive behaviors that contribute to stress-related disorders. Our previous work demonstrates that chronic unpredictable stress (CUS) induces a persistent increase in the firing activity of proopiomelanocortin (POMC) neurons in the arcuate nucleus (ARC). This hyperactivity is due, in part, to a reduction in GABAergic synaptic transmission onto POMC neurons, indicating a disruption in inhibitory control. However, the sources of GABAergic inputs responsible for this effect of chronic stress are unknown. Although AgRP neurons provide local GABAergic input onto POMC neurons and are suppressed by chronic stress, chemogenetic activation of AgRP neurons during stress exposure failed to reduce POMC neuron hyperactivity. GABAergic projections originating from the dorsomedial hypothalamus (DMH) represent another source of inhibitory input to POMC neurons. We found that CUS decreased the firing activity of DMH GABAergic neurons with sex differences, with females exhibiting greater vulnerability to stress-induced suppression. Chemogenetic activation of these neurons during chronic stress markedly attenuated POMC neuron hyperactivity in both sexes, indicating that DMH GABAergic neurons function as a critical upstream regulator of POMC neuron activity under chronic stress. These findings suggest that reduced inhibitory input from DMH GABAergic neurons, rather than local GABAergic AgRP neurons, drives POMC neuron hyperactivity. The weakening of the DMHGABA[->]ARCPOMC circuit activity may represent a novel mechanism underlying maladaptive stress responses and a potential therapeutic target for stress-related disorders.

The hypothalamic pituitary adrenal (HPA) axis is a critical regulator of physiologic and psychological responses to acute and chronic stressors. HPA axis function is control by numerous feedback inhibitory mechanisms, disruptions of which can lead to various psychiatric conditions, such as depression, posttraumatic stress disorder, and schizophrenia. Vagus nerve stimulation has been shown to be efficacious in the treatment of in these various mental health issues potentially via modulation of HPA axis function, but the mechanisms by which the vagus nerve may regulate HPA function has not been fully elucidated. In the present studies, we sought to test the hypothesis that the vagus nerve is a critical regulator of HPA function. Neuroendocrine function and neurocircuit changes in corticotropin releasing factor (CRF) neurons in the paraventricular nucleus of the hypothalamus (PVN) were examined following acute stress after subdiaphragmatic left vagotomy (VX) in adult male Sprague-Dawley rats. We found that VX mimics HPA activation seen in sham surgery animals exposed to acute restraint stress, particularly increased plasma corticosterone levels, elevated PVN CRF mRNA, and increased action potential firing of putative CRF neurons in PVN brain slices. Furthermore, VX animals exposed to acute restraint stress showed increased elevations of plasma corticosterone and PVN CRF mRNA which may be due to lack of compensatory PVN GABAergic signaling in response to acute stress. Both Sham/Stress and VX/no stress conditions increased action potential firing in putative PVN CRF neurons, but this effect was not seen in the VX/stress condition, suggesting that not all forms of stress compensation are lost following VX. Overall, these findings suggest that the vagus nerve may play a critical role in regulating HPA axis function via modulation of local PVN neurocircuit activity.